1. Field of the Invention
The present invention relates to a process for synthesizing a nucleic acid sequence, which is useful in the field of genetic engineering, as well as a process for amplifying it. More particularly, the present invention relates to a process for synthesizing a nucleic acid sequence with use of strand displacement reaction, and to a process for amplifying it.
2. Background Art
In the field of genetic engineering, there is known an assay based on the complementation of nucleic acids as a method which is capable of directly analyzing genetic features. In such assay, if an aimed gene is present only in a small amount in a sample, it is necessary to previously amplify the aimed gene itself generally due to the difficulty of its detection.
The amplification of the aimed gene (amplification of nucleic acid) is primarily carried out by enzymatic methods with use of DNA polymerase. Such enzymatic methods include, for example, the polymerase chain reaction method (PCR method; U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), and the reverse transcription PCR method which is the combination of the PCR method and the reverse transcriptase method (RT-PCR method; Trends in Biotechnology, 10, 146-152, 1992). These methods is intended to make capable of amplifying the aimed gene from DNA or RNA by repeating the three step reactions of the dissociation (denaturation) of a double-stranded nucleic acid as a template into a single-stranded nucleic acid, the annealing of a primer to the single-stranded nucleic acid and the synthesis (extension) of a complementary strand from a primer. These methods require the repetition of three steps in total in which the reaction solution is adjusted to a temperature suitable for each reaction in the three steps described above.
There is known the shuttle PCR method as an improvement in the amplification method of nucleic acids described above (“Recent Trends of the PCR method”, TANPAKUSHITSU KAKUSAN KOSO (Proteins, Nucleic Acids and Enzymes), KYORITSU SHUPPAN CO., LTD., Vol. 41(5), 425-428 (1996)). In the shuttle PCR method, two steps of the annealing of a primer and the extension among the three-step reactions in the PCR method are carried out at the same temperature, so that the aimed gene can be amplified by the reactions of two steps in total. Furthermore, EP Laid-Open Publication No. 0320308 discloses the ligase chain reaction method (LCR method), in which a known gene sequence is amplified by two-step temperature cycling (repeated reactions with heating and cooling).
In the methods described above, it is necessary to use a thermal cycler which can control temperature strictly in an extensive range. In addition, these reactions are carried out at two or three temperature conditions and require time for adjusting respective reaction temperatures, so that the more increased the cycles, the longer the time required for it.
In order to solve the aforementioned problems, methods for amplifying nucleic acids which can be conducted under an isothermal condition have been developed. Such methods include, for example, the strand displacement amplification (SDA) method and the self-sustained sequence replication (3SR) method described in Japanese Patent Publication No. 7/114718, the nucleic acid sequence based amplification (NASBA) method and the transcription-mediated amplification (TMA) method described in Japanese Patent No. 2650159, the Q beta replicase method described in Japanese Patent No. 2710159, a variety of the improved SDA methods described in U.S. Pat. No. 5,824,517, International Publications WO99/09211 or WO95/25180, the LAMP (Loop-Mediated Isothermal Amplification) method described in International Publication WO00/28082, the ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) method described in International Publication WO02/16639, and the like. The reactions of all steps involved in the isothermal amplification of nucleic acids proceed simultaneously in reaction mixtures maintained at a constant temperature.
In the SDA method, it is possible to amplify the aimed nucleic acid (and its complementary strand) in a sample by the displacement of a double strand mediated by DNA polymerases and restriction endonucleases. This method involves four primers, of which the two primers must be designed to include the recognition sites of the restriction endonucleases. In addition, this method requires as a substrate for the synthesis of nucleic acids modified deoxynucleotide triphosphates such as a deoxynucleotide triphosphate in which the oxygen atom of the phosphate group at the alpha-position of the triphosphate moiety has been substituted by a sulfur atom (S). This method requires high running cost. Furthermore, in this method, modified nucleotides such as alpha-S-displaced deoxynucleotides are included in the amplified nucleic acid fragment, so that when the amplified fragment is subjected to the restriction enzyme fragment length polymorphism (RFLP) assay, it cannot be broken with the restriction enzyme and thus such assay cannot be practiced in some cases.
The SDA method described in U.S. Pat. No. 5,824,517 requires a chimeric primer comprising RNA and DNA, in which DNA is in the 3′-end side. Such chimeric primer composed of RNA and DNA requires high cost of its synthesis, and RNA containing primers also require professional knowledge for its handling. In addition, the improved SDA method described in International Publication WO99/09211 requires a restriction enzyme which generates a 5′-protruding end, and the improved SDA method described in International Publication WO95/25180 requires at least two primer pairs, so that these methods also require high running cost.
The ICAN method requires a chimeric primer comprising RNA and DNA, in which RNA is in the 3′-end side, and an RNase H for cutting the RNA moiety at the 3′-end of the primer, so that reagents required for the method cost a great deal. Thus, this method requires high running cost particularly in genetic tests on a large amount of samples.
The LAMP method requiresfour primers, which recognize six regions to amplify the aimed genes. That is, in this method, the first primer anneals a template strand to cause extension, and a stem-loop structure is formed at the 5′-end portion of the extended strand due to the constitution of the first primer. The extended strand is next separated from the template strand by the strand displacement reaction of the second primer which is designed in the upper-stream of the first primer. Similar reactions occur repeatedly also on the other strand of the double-stranded nucleic acid, and thus the target nucleic acid is amplified. Therefore, the mechanism of the amplification reactions is complicated, and the six regions must be selected, so that it becomes difficult to design primers. In addition, the two of four primers are required to be comparatively long chain primers, and thus the synthesis and purification of the primers is expensive and takes a lot of time.
Therefore, there is a need for a process for amplifying nucleic acids, which can be practiced with a low running cost and the nucleic acid fragment thus obtained can be further used for genetic engineering treatments. Particularly, it is desired to have an isothermal nucleic acid amplification method in which amplification can be conducted quickly with a pair of primers.